Method for propagating yeasts and molds by mixed culturing and method of fermentation thereof

ABSTRACT

A strain of bacteria is added to a fermentation system wherein yeasts or molds capable of assimilating hydrocarbons or carbohydrates are fermented using hydrocarbons or carbohydrates as the main carbon source, and mixed culturing is carried out.

United States Patent [1 1 Miura Feb. 19, 1974 METHOD FOR PROPAGATINGYEASTS AND MOLDS BY MIXED CULTURING AND METHOD OF FERMENTATION THEREOF[76] Inventor: Yoshiharu Miura, 8-28,

Katagiri-cho, 1baragi-shi, Osaka, Japan [22] Filed: May 27, 1971 21Appl. No.: 147,422

[30] Foreign Application Priority Data UNITED STATES PATENTS 2,766,17610/1956 Jeffreys l95/111X 2,636,823 4/1953 de Becze 195/111 X 1,875,4019/1932 Woodruff el al. 195/111 X 3,536,586 10/1970 Lee et al 195/111 X3,667,968 6/1972 Kasik et a1. 195/111 X Primary Examiner-Alvin E.Tanenholtz Assistant ExaminerR0bert J. Warden Attorney, Agent, orFirm-Fitzpatrick, Cella, Harper & Scinto [5 7 ABSTRACT A strain ofbacteria is added to a fermentation system wherein yeasts or moldscapable of assimilating hydrocarbons or carbohydrates are fermentedusing hydrocarbons or carbohydrates as the main carbon source, and mixedculturing is carried out.

6 Claims, 4 Drawing Figures Pmmn im Y 3.793.153

SHEEI 1 Bf 4 FIG. 1A

2 Single n icroorganism culturing bovooq Strain D(yeast) 4"" StrainF(bacterium) Amount of cells formed (number of cells/ml.) o N 0 a 16 2432 40 4a 56 Time (hour) I NVEN TOR.

Yosh i han'u MTV-RA METHOD FOR PROPAGATING YEASTS AND MOLDS BY MIXEDCULTURING AND METHOD OF FERMENTATION THEREOF BACKGROUND OF THE INVENTIONThe present invention relates to a method for propagating microbialcells wherein yeasts or molds capable of assimilating hydrocarbons orcarbohydrates are fermented using hydrocarbons or carbohydrates as amain carbon source, and mixed culturing is carried out by addingbacteria to the fermentation system.

Great interest has recently developed in the use of hydrocarbons andcarbohydrates as the main carbon source in fermentation and assimilationprocesses. For example, considerable research has been directed towardthe utilization of unicellular protein obtained by propagatingmicroorganisms using petroleum hydrocarbons as the main carbon source,as well as the production of metabolic products such as, enzymes, aminoacids, vitamins, organic acids and other useful substances. However,research in this area for industrial purposes has been directed only tothe cultivation of a single pure strain of microorganism for a specificend product.

The principal considerations in industrial processes are the growth rateof the microorganisms in the culture and the yield of desired metabolicproducts. The present invention is, in part, based upon the finding thatmixed cultures of one or more species of assimilating microorganisms ina culture medium wherein the carbon source is a mixture of numeroushydrocarbons such as petroleum fermentation, the range of assimilatedcarbon source can be broadened, the rate of carbon conversion can beincreased, the kinds of metabolic products can be increased andscreening is facilitated.

SUMMARY OF THE INVENTION In the present invention, bacteria and molds oryeasts are subjected to mixed culturing in a medium containinghydrocarbons or carbohydrates as a carbon source and together with othernutrient sources necessary for the growth of the microorganisms.Although the optimum temperature for propragating bacteria is 36C. ascompared to 30C. for yeasts and molds, and the division period of yeastsor molds is considerably longer than that of the bacteria, the mixedculturing of the present invention has the advantage that the optimumtemperature for'propagating yeasts or molds is considerably elevated andapproaches that for propagating bacteria. Moreover, the rate ofpropagation is also accelerated, the range of assimilated carbon sourceis remarkably broadened, the yield of cells is increased and the kindsof metabolic products are increased.

The increase of the rate of fermentation and the rate of propagation dueto the rise of the optimum temperature for fermentation and the increaseof the yield of cells has a very important significance in the industry,because they directly relate to the increase of the yield rate ofunicellular protein or useful metabolic products.

The hydrocarbon and carbohydrate assimilable microorganisms which areuseful in the present invention are selected for example, as yeasts,from the families, Cryptococcaceae and Endomycetaceae; as bacteria, fromthe families Actinomycetaceae, Achromobacteraceae, Bacillaceae,Bacteriaceae, Micrococcaceae, Pseudomonadaceae, Mycobacteriaceae,Brevibacteriaceae and Corynebacteriaceae; and as molds, from the classAscomycetes.

DESCRIPTION Mixed culturing according to the present invention ispreferably carried out by fermentation, under aerobic conditions, of anaqueous nutrient media such as by shaking culture or stirring culture.On an industrial scale, it is most advantageous to employ theaerationstirring submerged culture technique.

Culturing is generally carried out at a temperature of 25 to 47C. and ata pH OF 3.5 to 8.5. However, when a high production of yeast is desired,it is preferred to adjust the pH to be about 3.5 to 5.5. In the samerespect, when a high production of bacteria or mold is desired it ispreferred to adjust the pH to be about 6.0 to 8.5. Culturing time isproperly determined by the strains of microorganism used, the mediumcomposition and the desired object of culturing. However, the culturingperiod is generally 8-120 hours.

As for the culture medium in the present invention, those having acomposition normally used in the ordinary culture of yeasts, molds andbacteria can be used. However, about I to 2 percent by volume ofhydrocarbons or carbohydrates based on the total amount of the mediumare added thereto as the carbon source. When small amounts ofwater-soluble vitamins, such as riboflavin, thiamine, p-aminobenzoicacid, pyridoxine hydrochloride, calcium pantothenate, nicotinic acid,biotin, folic acid, etc. are added to the medium, the growth of themicroorganisms is increased.

As hydrocarbons which can be used as the carbon source, gaseoushydrocarbons, naphtha, light oil, kerosene, liquid paraffin, heavy oil,etc. are appropriate. In addition, saturated and unsaturated aliphatic,alicyclic or aromatic compounds may be used. As the carbohydrates whichcan be used as the carbon source, glucose, glycerol, fructose, sucrose,maltose, mannose, mannitol, xylose, galactose, starch, starchhydrolyzate, molasses, etc. can be used.

As the nitrogen source, peptone, NZ amine, meat extract, yeast extract,corn steep liquor, soy bean powders, protein hydrolyzate, inorganicnitrates, ammonium salts, etc. can be used. Further, as inorganic salts,sodium chloride, calcium chloride, magnesium sulfate, calcium carbonate,phosphates, and small amounts of heavy metal salts such as iron, copper,manganese salts, etc. can be used. 1 7

Since hydrocarbons are almost insoluble in water, it is preferable, whenadded to the liquid medium, to vigorously stir the hydrocarbon with theaqueous solution to form a fine suspension or to add a suitablesuspending agent and dissolving agent.

In practice of the invention, two groups of the strains ofmicroorganisms are inoculated in the medium, but these two strains canbe precultured separately and then the resulting precultures can beinoculated in the medium at the same time. Alternatively, one of thestrains can be inoculated to the other cultured strain and then both canbe inoculated in the medium. Furthermore, the two strains ofmicroorganisms to be used may be subjected to mixed culturing in advanceand the resulting mixed culture can be inoculated in the mainfermentation medium. In any case, the ratio of the amounts of the twostrains to be inoculated can be changed as desired according to thepurpose of the cul- I ture. Generally, it is preferable that the two ormore strains are inoculated respectively at a ratio of a microorganismof 10 l cells/cc. and the main culturing is carried out. Moreover, thestrains of the two groups to be inoculated are not restricted to onlyone kind in the respective groups but more than one strain, that is, anydesired number of strains may be included in the respective groups, solong as they can meet the object of the mixed culture.

After the fermentation of the mixed culture is terminated, the metabolicend products may be separated from the culture liquid in any well knownmanner. For example, an amino acid may be separated from the mediumafter removal of the microorganism cells by means of an ion exchangeresin treatment.

Practice of certain specific embodiments of the invention areillustrated by the following examples:

EXAMPLE I In this example, Mycotorula japonica (ATCC 20311) (Strain D)is used as the yeast strain, and Bacillus subtilis (ATCC 21662) (StrainF) is used as the bacterium strain. Each of the strains is initiallyslant-cultured on potato-agar medium at 27C. for 24 hours. Thepotatoagar medium comprises 20 percent potato, 3 percent yeast extractand 1 percent hydrocarbon (mixture of l2 26v 1a 2s u so 15 329 and 1634)- In the case of carbohydrate-assimilable microorganisms, 1 percentglucose is used in place of the hydrocarbons.

After culturing, thestrains are respectively inoculated by one platinumloop into 500 cc. Erlenmyer flasks containing a basal medium comprising10 ml./l. hydrocarbons (mixture of n-C I-I n-C H n-C l-l n-c, H,, andn-C I-I 5.0 g./l. NI-l I-I PO 4.0 g./l. KH PO. 0.3 g./l. K d-IP0 0.4g./l. MgSO -7I-I O, 0.1 g./l. NaCl, 0.1 g./l. CaCl -2I-I O, 1X10 g./l.CuSO '5- H O, 4X10 g./l. FeCl -6I-l O, 8X10 g./l. MnSO '7- H O, 4X10g./l. NaMoO,-H O, 8X10 g./l. ZnSO -7- H 0, 1 ml./l. vitamin solution(200 mg. riboflavin, 100 mg. thiamine, 100 mg. p-aminobenzoic acid, 100mg. pyridoxine hydrochloride, 100 mg. calcium pantothenate, 100 mg.Nicotinic acid, 1 mg. biotin and 1 mg. folic acid in 1 liter) anddistilled water to adjust the volume to 1,000 ml.. In cases where themicroorganisms are carbohydrate-assimilable strains, 5 percent glucoseis used in place of the hydrocarbons.

The above cultures are then precultured at a temper ature of 30C.withrespect to the yeast and, 46C. with respect to the bacterium for'48hours with shaking at an amplitude of 5 cm. and 100 reciprocations perminute. The thus obtained precultures are respectively centrifuged at3,000 revolutions per minute for 10 minutes to collectthe microbialcells which are thereafter washed with a 0.1 M'phosphate bufier solution(pH 7.0).

The washed cells are then respectively inoculated into a basal medium ofthe same composition as given above in such manner so that the culturescontain 10 10" cells/cc. Fermentation of the main culture is carried outwith shaking and thereafter the cells are counted under a microscopewith a Thomas blood cell counter for the yeast strain employed and witha bacteria counter chamber for the bacterium strain employed. For themixed culture, the number of respective microbial cellsare counted undera microscope in M oldsi K: Periicilliurh jahthihel idrh TIFIGWEVZAICC InFIG. 1A, the bacterium strain A and yeast strainoare cultivatedseparately. After 48 hours, both strains reach "maximum cell growth.However, as shown in FIG. 13, maximum cell concentration occurs at about38 hours with both strains plateauing at about the same time.

When the single cultures are cultured at 42C. as is shown in FIG. 2A,maximum concentration of the yeast strain occurs after about 40 hours ofculturing. Moreover, propagation is not great. However, when culturingis mixed in accordance with the present invention, the cultivation timeand degree of propagation is greatly enhanced with respect to bothstrains. FIG. 2B shows the results of mixed culturing at 42C.. As isevident from the FIG. 2B, both strains exhibit maximum propagation afteronly about 16 hours of cultivation. In addition, both strains propagateto a much greater extent than in single culturing.

It is evident in this case from these results that the rate ofpropagation is accelerated and the optimum temperature for propagatingis considerably elevated by mixed culturing.

EXAMPLE n In this example, the following microorganisms are cultivated:

B: Torulopsis dattila I.F.O. 0662 (ATCC 20309) C: Rhodotorula glutinisI.F.O. 0388 (ATCC 20310) D: Mycotorula japonica I.A.M. 4185 (ATCC 20311)Bacteria:

Nb'cbrdi coral li na I.F.O. 3358 C(ATC621664) i Bacillus sublilis JB15(ATCC 21663) G: Micrococcus cerificans I.F.O.

21662) H: Pseudomonas aeruginosa I.F.O. 3923 (ATCC 21661) 7 I:Corynebacterium hydrocarboclastus (ATCC 6946) J: Brevibacteriumacetylicum I.F.O. 12146 (ATCC Cultivatioifis cam" "ea an? iiimesasraarasea in Example I except that the cultivation temperature isvaried and the carbon source is changed in accordance with themicroorganisms used. The variables, percent- 12552 (ATCC ages of yieldsof the cells and the maximum specific microbial cells are shown in thefollowing Tables I and 'growth rates calculate on the basis of thenumbers of II:

TABLE 1 (carbon maize? hydrocarbons) Microorganisms CulluringHydrocarbons Yield of cells (70) Maximum specific Useful products usedtemperature used (mixture) growth rate (hour"') D 30C. n-alkanes of 75.60.l24 Aspar tic acid G lutarnic acid, Leucine,

w Serine, Histidine, Lysine F 46C. n-alkancs of 67.3 0.30l Dipicolinicacid, Aspartic acid, Glutamic CI2 CIS acid, Alamin e, LeucineTArginine,Lysine D F 42C. n-alkancs of 90.3 D=0.34l F=0.347 Aspartic acid Glutarnic il Lt cir c m I2 l6 Serine, Histidine, Lysine, Arginine, AlanineA 30C. n alkanes of 74.2 0.131 Oil and fat, Citric acid, Aspartic acid,

Cn-Cw Glutamic acid, Leucine, Histidine E 4IC. n-alkancs of 68.1 0.292Fal, Lysine, Diaminopimelic acid,

Homoserine, Ornithine, Phenylalanine,

Tyrosine, Citrulline, Polycarboxylic acids A E 32C. n-alkanes of 87.]A=0.235 e=0.242 Fat, Oiland fail;itric fifhglutamicacid,

C W Aspartic aciEhLysice, Leucine,

Diaminopimelic acid, Homoserine, Ornithine, Phenylalanine, Tyrosine,Citrulline, Polycarboxylic acids A F 37C. n-alkancs of 98.8 A=0.346F=0.367 Oil and fat, Citricacid Glulamic acid,

Aspartic acid, Leucine, Arginine, Lysine G 40C. n-alkanes of 69.3 0.253Alanine, Valine, Aspartic acid, Glutamic acid, Leucine, Arginine, LysineA G 35C. n'alkanes of 94.5 A=0.306 G=0.3ll Oil and fat, Citric acid,Glutamic acid,

Aspanic acid, Leucine, AFginine, Lysine H 37C. n-alkanes of 72.9 0.29Salicyclic acid,Eiotin elatedsubstzmces,

Cs-Cw Catechol, Aspartic acid, Leucine,

lsoleucinc, Dipicolinic acid A H 37C. n-alkanes of 96.8 A=0.326 H=0.34lOil and fat, Citric acid, Glutamic acid,

CHI-C16 Aspartic acid, Leucine, Arginine, Lysine l 38C. n-alkanes of72.l 0.23 Glutan ic acid, A lanine, Yaline, (jlycjge, V

CII CH Lysine, Diaminopimelic acid, Homoserine,

Ornithine, Citrulline, Tyrosine, Phenylalanine, Polycarboxylic acids i36 E? ]i-a|i ari; s i 35.1 A=6fi5 j i=63if91 g Tcim @155, mi PM,Glutamic acid, Aspartic acid, Leucine 1 28C. n-alkancs of 7 l .6 0.152Glutamic acid, Aspartic acid, Leucine CIFCM Lysine, Leucine A 32C.n-alkancs of 76.6 A=0.l8l J=O.l93 Oil and fat, Citric acid, Glutamicacid,

Aspartlc acid, Arginine, Lysine, Leucine B 30C. n-alkanes of 73.7 0.125Aspartic acid, Glutamic acid, Leucine,

CIFCW Histidin e B E 329C. n-alkanes of 85.8 B=0.235 E=0.246 Glutamicacid, Aspartic acid, Leucine,

lo l! Histidine, Lysine, Diaminopimelic acid,

Homoserine, Ornithine B F 37C. n-alkancs of 92.4 B=0.332 F=0.349Glutamic acid, Aspartic acid, Leucine,

C ill M Histidine, Arginine, Lysine B G 35C. n-alkanes of 90.2 B=O.278G=0.293 Glutamic acid, Aspartic acid, Leucine,

CWCW Histidine, ArgininefL ysine B H 37C. n-ulkanes of 91.2 B=0.3l3H=0.325 Glutamic acid, Aspartic acid, Leucine,

CW'CW Histidine, Arginine, Lysine B l 37C. n-alkanes of 9l.3 B=0.304l=0.324 Vitamin B11. Glutamic acid, Aspartic acid,

Leucine, Histidine C 28C. n-alkanes of 75.l 0.! l7 Asparlic acid,Glutamic acid, Leucine,

CwCn Histidine C E 32C. n-alkancs of 85.2 C=0.2l3 E=0.249 Glulamie acid,Aspartic acid, Leucine,

Cw-Cw Histidine, Diarninopimelic aad,

Homoserine, Ornithine C F 37C. n-alkancs of 96.8 C=0.338 F=0.354Glutamic acid, Aspartic acid Leucine, cwcw Histidine, Lysine,SerinejArgihiiie C G 35C. n-alkancs of 94.5 C=0.302 6 03 17 Glutamicacid, Asparlic acid Lcucine, I

ow Histidine, Arginine, Lysine 7 i TABLE 1 cariiaflsauresi hydrocarbons)Microorganisms Culturing Hydrocarbons Yield of cells Maximum specificUseful products used temperature used (mixture) growth rate (hour") C H37C. n-alkancs of 96.4 C=0.329 H=0.334 Glutamic acid, A spartic I l0 lflHistidine, Arginine, Lysine C l 36C. n-alkancs of 96.4 C=0.328 l=0.337 Glu tam i c acid Aspartic aci d, Le u cinc, V V

CHI-CIR Histidine, Vitamin B12 V D E 32C. n-alkancs of 86.3 D=0,232E=0.249 Glutamic acid, Aspartic acid, Leucine,

C w Lysine, Diamifiopfiielimid,Homoserine,

Ornithine D F 37C. n-alkancs of 98.] D=0.352 F=().369 Glutamic acid,Aspartic acid, Lcucine,

xiim Lysine. Serine. Argininc l) (i 36"(1. n-alkuncu of 92.8 [M0296(l='().3l3 (ilutnmic ucitl. Anpartlc ncld, Lcucinc,

lo IR Lysine, Arginine D H 38C. n-alkunes of 97.1 D=0.33l H=0.344Glutamic acid, Aspartic acid, Leucine,

CWCW Lysine, Arginine 7 7 D I 35C. n-alkancs of 96.8 D=0.329 l=0.334Gluta mic acid Asp artic acid, Lcucine A lolo Lysine, Vitamin B12 B .I32C. n-alkanes of 76.] M

ID l C J 32C. n-alkancs of 74.7 C=O.l44 J=0.l68 Glutarnic acid, Asparticacid, Le u cine,

CHI-C" l-listidine, Arginine, Lysine D +1 32C. nalkanes of 75.3 D=0.l72J=0.l85 Glutamic acid Aspartic acid, Arginine,

CID-C16 Lysine, Leucine K 28C. n-alkancs of 75.3 O.l5l Citric Acid HI- mK I 37C. n-alkanes of 937 K=0.29l |=0.289 Phenylalanine, Polycarboxylicacids, Citric C5-C1" acid,Glutamic acid, Alanine, Valine,

Glycine, Lysine, Diaminopimelic acid, Homoserine, Ornithine, Citrulline,Tyrosine L 28C. n-alkanes of 74.7 0.143 Amylase ir in L E 40C. n-alkanesof 93.2 L=0.305 E=0.3l l Amylase, Fat, Lysine gialninopimelk: acid,

5 \a Homoserine, Ornithine, Phenylalanine,

Tyrosine, Citrulline, Polycarboxylic acids TABLE 2 (carbon sources:carbohydrates) Microorganisms I Culturing Carbon source Yield of cellsMaximum specific Useful products used temperature growth rate (hour D30C. Glucose 81.7 0.204 Aspartic acid, Glutamic acid, Leucine,

Histidine F 45C. Glucose 83.8 0 .3l2 Aspartic acid, Glutamic acid,Alanine,

Leucinc, Argininc, Lysine D F 41C. Glucose 95.6 D=0.36l F=0.364 Asparticacid, Glutamic acid, Alanine,

Lcucine, Histidine, Argininc, Lysine K 28C. Glucose 82.6 0.2l1 Citricacid v r l 37C. Glucose 83.3 0.327 Glutamicacid, Alanine, Valine,Glycine,

Lysine K l 37C. Glucose 96.8 K=0.372 I=0.381 Glutamic acid, Alanine,Valine, Citric acid,

Lysine, Glycine EXAMPLE iii" In this example, the followingmicroorganisms are cultivated in the same manner as Example ll:

Yeasts 77 (Y i) came 'zro'pibaiis (Y 2) Candida intermedia (Y 3 Candidalipolytica (Y 4) Candida albicans (Y 5) Candida rugosa (Y 6) Candidapetrophilum (Y 7) Candida brumptii (Y 8) Candida catenulata (Y 9)Candida melinii (Y l) Candida parapsilasis (Y 11) Candida pulcherrima (Y12) Candida reukaufii (Y 13) Candida cloacae TABLE 3 -Continued Singleculturing (ycust) (carbon sources: hydrocarbons) (Y 14) Candida maltosa(Y 15) Candida tenuis (Y 16) Saccharomyceteae pichia guilliermondii (Y17) Saccharomyceteae pichia farinasa (Y 18) Saccharomyceteae pichia vini(Y l9) Saccharomyceteae debaryomyces klosckeri (Y 20) Saccharomyceteaedebaryomyces hansenii (Y 21) Saccharomyceteae debaryomyces vanriji (Y22) Torulopsis petrophylum (Y 23) Torulopsis dattila (Y 24) Torulopsisfamata (Y 25) Torulopsis sake (Y 26) Rhodotorula rubra (Y 27)Rhodotarula glutinis (Y 28) Rhodotorula gracilis (Y 29) Rhodotorulamucilaginosa (Y 30) Saccharomyceteae hansenula anonala (Y 31)Saccharomyceteae mycotorula japonica (Y 32) Trichosporon capitatum (Y33) Trichosporon japonicum (Y 34) Brettanomyces lambicus Bacteria:

(B 1) Pseudomonas aeruginosa (B 2) Pseudomonas oleovoran (B 3)Pseudomonas ovalis (B 4) Micrococcus cerificans (B 5) Micrococcusparaffinae (B 6) Micrococcus glutamicus (B 7) M ycobacterium smegmatis(B 8) Mycobacterium lacticolum (B 9) M ycobacterium album (B 10)Mycobacterium rubrum (B 11) Mycobacterium paraflinicum (B 12)Corynebacterium petrophilum (B l3) Corynebacterium simplex (B 14)Corynebacterium hydrocarboclastus (B l5) Corynebacterium brevicale (Bl6) Corynebacterium aleophilus (B 17) Nocardia opaca (B 18) Bacteriumaliphaticum (B 19) Bacillus stearothermophilus (B 20) Bacillus subtilis(B 21) Achromobacter cycloclastes (B 22) Achromobacter pestifer (B 23)Achrombacter delmarvae (B 24) Nocardia gardneri (B 25) Nocardiacorallina (B 26) Brevibacterium guale After completion of thefermentation period, the cells are counted in the same manner as in theabove Examples and growth rate is calculated therefrom. The results forsingle and mixed culturing are given in the following Tables 3, 4 and 5.

TABLE 3 Single culturing (yeast) (carbon sources: hydrocarbons)Microorgan- Culturing Yield of cells Maximum 5 ism used temperature[cells specific growth (g)/substrate rate (hour') (Y-S) 28-30 0.7330.117 (Y-6) 27-30 0.822 0.282 (Y-7) 25-28 0.634 0.043 (Y-8) 25-27 0.6610.0615 10 (Y-9) 27-30 0.612 0.041 (Y-10) 28-30 0.737 0.162 (Y-l 1) 28-300.745 0.174 (Y-12) 27-29 0.711 0.087 (Y-13) 25-28 0.707 0.076 (Y-14)26-29 0.71 0.081 (Y-) 26-30 0.75 0.082 15 (Y-l6) 2140 0.875 0.293 (Y-17)27-30 0.724 0.122 (Y-l 8) 26-30 0.656 0.0608 (Y-19) 26-30 0.748 0.114(Y-) 26-30 0.913 0.310 (Y-21) -28 0.641 0.0591 (Y-22) 27-30 0.776 0.21520 123 28-30 0.137 0.125 (Y-24) 28-30 0.747 0.145 (Y-25) 25-27 06280.0573 (Y-26) 26-29 0.731 0.113 (Y-27) 26-30 0.751 0.117 (Y-28) 27-300.748 0.126 (I-29) 26-29 0.735 0.111 25 (Y-30) 27-30 0.741 0.121 (Y-31)27-30 0.756 0.124 (Y-32) 27-30 0.739 0.119 (Y-33) 26-29 0.757 0.133(Y-34) 26-28 0.736 0.114

TABLE 4 Single culturing (bacterium) (carbon sources: hydrocarbons)Microorgan- Culturing Yield of cells Maximum 35 ism used temperature(cells specific growth (g)/substrate rate (hour") (B-l) 37-40 0.729 0.29(B-2) 37 0.748 0.281 (B-3) 36 0.751 0.277 40 (B-4) 33-40 0.693 0.253(13-5) 33-37 0.787 0.285 (B-6) 32-36 0.751 0.274 (B-7) 36-40 0.814 0.293(B-8) 35-40 0.808 0.289 (B-9) 34-38 0.797 0.283 (13-10) 34-37 0.7880.278 (B-11) 33-37 0.783 0.275 (8-12) 30-35 0.738 0.266 (13-1 3) 30-370.784 0.289 (B-l4) 30-37 0.793 0.294 (8-15) 30-37 0.787 0.291 (B-16)30-37 0781 0.285 (B-17) 30-32 0.733 0.247 (8-18) 35-42 0.684 0.283(13-19) 50-60 0.663 0.158 (8-20) 33-40 0.805 0.311 (13-21) 30-36 0.7330.261 (B-22) 25-32 0.726 0.183 (8-23) 26-32 0.733 0.191 (13-24) 25-320.742 0.217 (13-25) 25-32 0.748 0.223 (13-26) 28-32 0.737 0.204

TABLE 5 Mixed culturing (carbon sources: hydrocarbons) MicroorganismCulturing Yield of cells Maximum used temperature [cells specific growth(g)lsubstrate rate (hour) (Y-2)+(B-1) 35-37 0.945 (Y-2)=0.311

(Y-3 )+(B-1) 35-37 0.968 (Y-3 )=0.326

TABLE 5 Continued Mixed culturing (carbon sourccx: hydrocarbons) TABLE 5-Continued Mixed culturing (carbon sources: hydrocarbons) TABLE 5Continued Mixed culturing (carbon sources: hydrocarbons) TABLE 5-Continucd Mixed culturing (carbon sources: hydrocarbons) TABLE 5Continued Mixed culturing (carbon sources: hydrocarbons)

2. A process according to claim 1, wherein said yeast strain belongs tothe family Cryptococcaceae or Endomycetaceae.
 3. A process according toclaim 1 wherein said carbon source is hydrocarbon.
 4. A processaccording to claim 1 wherein said carbon source is carbohydrate.
 5. Aprocess according to claim 1 wherein said culturing temperature is from25* to 47*C.
 6. A process according to claim 1 wherein the pH of saidnutrient medium is adjusted to from 3.5 to 5.5.